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Li et al. Microbiome Res Rep 2024;3:26 https://dx.doi.org/10.20517/mrr.2023.57 Page 7 of 16

was added to each well and was incubated at room temperature in the dark for 40 min. Finally, 1,000 μL 100
mM TrisHCl buffer containing freshly prepared 2 μg/mL trypsin (trypsin:proteins = 1:50) was added into
each well before being incubated at 37 °C, 800 rpm overnight in ThermoMixers.

Desalting
After digestion, desalting of protein lysate was performed using an automated liquid handler (Hamilton
Nimbus 96): first, each sample was acidified with 100 μL 10% FA to reach a pH of 2-3. The reverse-phase
(RP) desalting columns (e.g., IMCS, 04T-H6R05-1-10-96, 04T-H6R52-1-10-8 or equivalent) was
conditioned by two cycles of up-and-down mixing in 100% ACN and two cycles of mixing in 0.1% FA. The
recommended volume of mixing is 500-800 μL in each cycle. Next, samples were loaded to the pre-activated
reverse-phase (RP) columns by at least ten cycles of up-and-down mixing of 500-800 μL volume. The RP
columns were then washed by two cycles of up-and-down mixing in a first sample washing plate containing
0.1% FA, followed by being mixed in a second sample washing plate of 0.1% FA for another two cycles.

800 μL 80% ACN + 0.1% FA was transferred from the reservoir plate to the elution plate, and samples in the
RP columns were eluted by two cycles of 800 μL up-and-down mixing in the elution plate. From the elution
plate, 240 μL of the eluted solution was aliquoted to another 96-well plate to be used for TMT labeling. A
mixture of all sample aliquots is recommended to be used as the reference sample for the TMT labeling.
Samples were dried in a SpeedVac with a plate adapter at room temperature (check every 20 min until
samples are dried).

TMT-labeling and desalting
20 μL 100 mM TEAB in 20% ACN solution was added to each sample well and mixed sufficiently using
600 rpm on an orbital shaker. 15 μL mixture was aliquoted from each sample well to the corresponding
wells of the TMT reagent plates. The plate was covered with plate lids and incubated in the thermomixers at
25 °C, 600 rpm for 2 h. Then, 15 μL quencher (0.8% hydroxylamine in 100 mM TEAB) was added to each
well and reacted in the thermomixers at 25 °C, 600 rpm for 15 min. Next, the samples were acidified by
TM
adding 60 μL 5% FA to each well, followed by combining each set of TMT11plex by taking 80 μL from
each sample. Finally, all samples of a same row were combined into a 96-deepwell plate. Samples were
desalted and dried in a SpeedVac at room temperature.

LC-MS/MS analysis
TMT quantitation was performed using a high-resolution LC-MS/MS. Here, we used an UltiMate 3000
RSLCnano system coupled with an Orbitrap Exploris 480 mass spectrometer system; their setups are as
shown in Supplementary Tables 3 and 4, respectively. Samples were resuspended at 1 μg/μL protein in 0.1%
FA. After being sufficiently mixed using a vortex mixer, samples were centrifuged at 14,000 g for 5 min
before being loaded to a LC-MS/MS sampler plate. 1-2 μL of each sample was injected to the LC-MS/MS
and was analyzed following a 2-hours gradient.

Database search and data analysis
A database search of the LC-MS/MS raw files was performed using MetaLab 2.3. The software can be freely
downloaded at http://imetalab.ca. Here we used MaxQuant for a closed database search. The IGC database
was used as the microbiome protein FASTA database. Under the “Parameters” tab, “Carbamidomethyl (C)”
as fixed modifications, and “Acetyl (Protein N-term)” and “Oxidation (M)” as variable modifications were
selected. “Isobaric labeling” quantification mode of TMT11plex was selected. We used default MaxQuant
TM
search parameters pre-defined in MetaLab. Data pre-processing was performed using the MSstatsTMT R
package based on proteinGroups.txt, evidence.txt tables, and a user-customized msstatstmt_annotation file
as the inputs. The processed data table can then be used for downstream data analyses of principal